1. Field of the Invention
The field of this invention is the determination of phosphorylation of tyrosine on a receptor tyrosine kinase and screening of compounds that affect the phosphorylation.
2. Background
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. These materials may be consulted for specific language, which may be omitted from the present specification and are, as stated in the Conclusion, incorporated by reference. The discussion below should not be construed as an admission as to the relevance of the information to the appended claims or the prior art effect of the material described.
Pharmaceutical small molecule drug discovery is predicated on discovering compounds that bind to receptors or cytosolic proteins and act as agonists, antagonists, inverse agonists or modulators. One important class of proteins known as receptor tyrosine kinases (“RTKs”) are attractive targets, as these proteins act to induce a number of disease associated pathways. An important focus of pharmaceutical drug discovery is the identification of surrogate ligands for proteins, e.g., receptors, kinases, or other proteins in the pathway of phosphorylation. Of particular interest in this respect is a subclass of cell surface receptor proteins known as receptor tyrosine kinases. Another important class of proteins is the cytosolic kinases, which can phosphorylate one or a plurality of RTKs. By activating or inhibiting these kinases, one can inhibit the activation of the RTK target of the cytosolic kinase.
The RTK family functions in the regulation of cell growth, cell differentiation, adhesion, migration and apoptosis (Blume-Jensen and Hunter 2001 Nature 411:355-65) (Ullrich and Schlessinger 1990 Cell 61:203-12) (Schlessinger 2000 Cell 103:211-25) (Hubbard and Till 2000 Annu Rev Biochem 69:373-98). A number of human diseases have been linked to alterations in RTKs (Akin and Metcalfe 2004 J Allergy Clin Immunol 114:13-9) (Verheul and Pinedo 2003 Drugs Today (Barc) 39 Suppl C: 81-93) (Corfas et al., 2004 Nat Neurosci 7:575-80). Many RTKs have been identified as oncogenes in transforming retrovirus or human cancers (Hunter 2000 Cell 100:113-27) (Shawver et al., 2002) (Muller-Tidow et al., 2004), and recent reports indicate that RTKs may play a critical role in almost all types of human cancer (Shawver et al., 2002 Cancer Cell 1:117-23) (Prenzel et al., 2000 Breast Cancer Res 2:184-90) (Mass 2004 Int J Radiat Oncol Biol Phys 58:932-40). Both naturally occurring and artificial ligands that modulate RTK activity and signaling thus would be of tremendous interest from a therapeutic standpoint with respect to cancer and other diseases. (Haluska and Adjei 2001 Curr Opin Investig Drugs 2:280-6) (Sawyer et al., 2003 BioTechniques Suppl:2-10, 12-5). The ability to quickly, efficiently, and effectively screen vast libraries of compounds for particular activities has become a goal of the pharmaceutical industry. Desirably, the methods provide more than just binding information, frequently employing whole cells, where biological processes occur in relation to the compounds being screened.
Many cytokine receptors do not possess intrinsic kinase activity. However, they also initiate intracellular cascades of tyrosine phosphorylation. To do this they interact with separate proteins that are in the cytosol termed non-receptor tyrosine kinases (NRTK's). These proteins, such as the JAK kinases, bind to the intracellular domain of cytokine receptors. Once the cytokine receptor binds ligand and oligomerizes, this brings the JAK proteins into close proximity initiating trans-phosphorylation (by the JAK proteins) of the JAK proteins and the associated receptor.
High throughput screening has become a commonly employed strategy to identify novel compounds with particular activities from a diverse chemical library of compounds. Often, high throughput screening assays are either based upon measuring compound binding to defined molecular targets or measuring functional outputs resulting from compound/receptor interactions. However, both binding assays and functional assays have limitations. For example, for various technical reasons, binding assays are preformed in non-physiological conditions. Artificial, non-physiological conditions may impact and influence receptor pharmacology, leading to increased unreliability and difficulty in accurate interpretation of the data. Another drawback arises from the nature of the assay, which measures receptor binding only. Thus, binding competition assays do not provide information regarding the physiological function of ligands, such as whether the ligand functions as an agonist or antagonist. Since the only information obtained is binding, where the binding need not be at the target site, there can be many false positives.
Functional assays overcome many of the limitations associated with binding competition assays. Normally, cells are employed, which have the capability to respond to agonist binding as part of the assay protocol Therefore, the assays can provide a measure of the activity resulting from binding and allow for activity/concentration determinations. With the assay being performed under physiological conditions within the cell, one obtains results that more closely approximate the results that may be anticipated in vivo.
Several functional assays have been described for receptor tyrosine kinases. Exemplary assays include the quantification of autophosphorylation of RTKs (Olive 2004 Expert Rev Proteomics 1:327-41), measurement of phosphorylation of RTKs and downstream signaling molecules (ibid), measurement of intracellular calcium release (Dupriez et al., 2002 Receptors Channels 8:319-30), or measurement of RTK dependent cell proliferation (Mosmann 1983 J Immunol Methods 65:55-63) (Bellamy 1992 Drugs 44; 690-708). Despite the substantial variety of assays that have been developed for evaluating ligands for RTKs, there is still a substantial need for additional assays that can provide advantages as to the nature of the protocol, the involvement of the technician in performing the assay, the number of steps that can lead to errors in the result, the choice of equipment, the effect of organic solvents, the dynamic range and the sensitivity of the assay.
Relevant Patent Literature
U.S. patents and applications include U.S. Pat. Nos. 5,667,980; 5,773,237; 5,976,893; a group of patents with the same disclosure U.S. Pat. Nos. 5,891,650, 5,914,237, 6,025,145, and 6,287,784; 6,413,730, 2004/0038298, 2004/0161787; 2006/0199226; and 2008/0103107.